This is a SBIR Phase II application for developing microfluidic microchips containing 30k protein kinase (PK) substrate peptides for proteomic PK profiling of cell lysates. In recent years there has been a dramatic expansion in our understanding of PK (kinome) biology.1-7 In addition to the fundamental role of PKs in protein post-translational modification and cell signal transduction, PK enzymes have been associated with various forms of human cell growth malfunctioning and more recently with hematopoietic stem cell diseases.8-10 In fact, more than 200 PKs have been linked to human disease loci4, making PKs an important group of potential therapeutic targets. The success of the kinase-targeting therapeutic strategies has stimulated intensive efforts in new target identification and drug discoveries.11-14 A great need in these studies is to be able to effectively monitor the changes in PK levels under different cellular conditions. To realize the full potential of these studies, it will be vital for assays to have the capability of simultaneously monitoring the presence and quantitative levels of multiple PKs, not just a single kinase assayed per reaction. Such quantitative measures will allow for more accurate and reliable profiling of PKs on a proteome- scale. However, presently only limited choices for PK assays are available and these are mostly low throughput or only for qualitative assessments;the technologies for massively parallel PK profiling are still in their early stages, as there are major challenges and barriers to overcome. This proposed project seeks to build on the success of the Phase I project to develop a robust, quantitative, comprehensive, flexible, high throughput, cost effective and scalable PK profiling peptide chip platform. This will be a significant step towards addressing the rising needs of life science research and applications in the functional kinome fields. PUBLIC HEALTH RELEVANCE: Currently more than 200 protein kinases (PKs) have been associated with human disease, and the tremendous success of kinase-targeting therapeutic strategies has stimulated tremendous efforts toward identifying new kinase targets and potential drug discoveries. Unfortunately these efforts have been hampered by a lack of sensitive, quantitative assays for monitoring levels of multiple PKs simultaneously. The proposed 30k substrate peptide array aims to address this need, and allow highly accurate and reliable profiles of cellular kinase activity under different conditions, and has significant potential as a future diagnostic and/or drug screening platform.